A correlative electron microscopic and freeze-fracture examination of cat corneal endothelial wound repair.

In an attempt to create a model for sustained corneal edema in humans, the present study has examined wounded cat corneal endothelium. Small central (7 mm) wounds or large 90 percent debridement wounds were created with an olive tipped cannula and corneas sampled from 1 to 75 days post-wounding were processed for light and transmission electron microscopy and freeze-fracture. In small wounds, wound closure was complete by 14 days and corneal edema was absent. During wound closure, leading edge cell membranes had decreased intramembrane particles, numerous vesicle fusion sites and lacked cell junctions. Endothelium behind the wound margin was multilayered with fragmented cell junctions. After wound closure, endothelium returned to the morphology of non-wounded endothelium except that an abnormal posterior collagenous layer (PCL) was present. Wound closure was greatly retarded in large wounds and corneas remained edematous at 75 days. The morphology of the endothelial cells was similar to that in small wounds except for the presence of large multinucleated cells and a thicker PCL. These large wound findings are similar to those observed in chronically stressed dysfunctional human corneal endothelium and in this animal model may represent a similar response to injury.

Examination of sialic acid binding on dystrophic and normal retinal pigment epithelium.

In order to study differences in cell-surface sugars which may be involved in the phagocytic defect in Royal College of Surgeons (RCS) retinas, we have examined the presence or absence of lectin binding to carbohydrates on retinal pigment epithelial (RPE) plasma membranes of dystrophic (RCS-p+) and normal (Long-Evans) rats. A lectin which binds to both sialic acid and N-acetylglucosamine sugar residues, wheat germ agglutinin-ferritin (WGA-fe), was used. The specificity of WGA-fe binding to each sugar was studied by either pre-treating the tissue with neuraminidase enzyme which removes sialic acid residues, or by incubating the WGA-fe lectin with one of the haptens, N-acetylglucosamine. In non-enzyme-treated tissue, RPE cell-surface membranes from RCS retinas were densely labeled with WGA-fe as compared with the labeling on normal RPE, which appeared less dense and patchy in distribution. Wheat germ agglutinin-ferritin labeling in the presence of N-acetylglucosamine was blocked on both RCS and normal RPE surface membranes. After pre-treatment with neuraminidase, WGA-fe labeling on dystrophic RPE membranes was similar to non-enzyme-treated tissue but was enhanced on normal RPE. Labeling was blocked when N-acetylglucosamine was present with the lectin after enzyme pre-treatment. Other lectins which specifically bind to sialic acid, Limulus polyphemus agglutinin-ferritin (LPA-fe) and Limax flavus agglutinin (LFA) demonstrated sparse or no labeling on both RCS and normal RPE membranes. Our data suggests that N-acetylglucosamine residues predominate on RCS and normal RPE cell-surface membranes and that sialic acid binding sites are either not accessible to the lectins or may not be present.

Electron microscopy of corneal surface microdiathermy.

Microdiathermy has recently been shown to be effective in the treatment of persistent corneal epithelial recurrent erosion. In order to determine the mechanism of action of microdiathermy on the anterior surface of the cornea, rabbit eyes were treated with microdiathermy and the course of corneal tissue repair studied by electron microscopy. Shortly after treatment, the epithelium is edematous and necrotic and the lamina densa is thickened. Within 24 hours, the epithelium appears healed and some hemidesmosomes are present, but the lamina densa is still thickened. At two weeks the epithelial surface is undulated and protrudes into the stroma in areas where the lamina densa is disrupted. Hemidesmosomes are absent in these regions. Activated fibroblasts are present in superficial stroma. At four weeks following microdiathermy, there is segmental deposition of new lamina densa and a connective tissue zone between the newly deposited lamina densa and the old lamina densa. Hemidesmosomes are present only in areas of newly deposited lamina densa. Between six weeks and three months the epithelial basal surface becomes more uniform with mature hemidesmosomes and the new lamina densa is complete. The old lamina densa remains below it but is no longer present by six months. The mechanism of action for microdiathermy in recurrent erosion is believed to be as follows: in the treated area, epithelium and activated fibroblasts secrete a new connective tissue layer, which provides a suitable substrate to which the epithelium can adhere until it secretes a new lamina densa and hemidesmosome formation can occur.

Lectin-ferritin binding on dystrophic and normal retinal pigment epithelial membranes.

Phagocytosis in the rat retina is a process which involves uptake of shed photoreceptor outer segments by the overlying retinal pigment epithelium (RPE). In rats with inherited retinal degeneration, there is a defect in phagocytosis. One aspect of this phagocytic defect may be an alteration in glycoconjugate-containing membrane components on RPE membranes which mediate this phagocytic interaction. Lectin binding sites have been studied in order to localize the distribution of glycoconjugates on pigment epithelial microvilli in normal and dystrophic retinas and to determine if there are differences in the dystrophic retinas which would provide a clue about the defect. The following ferritin-conjugated lectins were used in this study: Concanavalin A (Con A-fe) or lens culinaris haemagglutinin (LcH-fe) for mannosyl-containing glycoconjugates; and wheat germ agglutinin (WGA-fe) for n-acetylglucosamine and sialic acid-containing glycoconjugates. Control tissue was incubated in the lectin in the presence of its competitor sugar. The number of ferritin particles or lectin-ferritin binding sites per micrometre of microvillous membrane was quantified from electron micrographs using a computer and a digitizing tablet. The number of WGA-fe binding sites on normal RPE microvillous membranes (56.0/micron) was statistically equivalent to the dystrophic membranes (48.8/micron). The number of Con A-fe binding sites on normal (27.3/m) and dystrophic RPE (26.7/micron) was also the same. A dramatic difference in LcH-fe binding sites was demonstrated on normal (1.5/micron) as compared to dystrophic RPE (19.1/micron). Our results indicate that more mannosyl residues are accessible on dystrophic microvillous membranes and, based on what is currently known about LcH binding, that these residues belong to glycoconjugates having fucosyl-containing carbohydrate cores. The data also suggest that in normal animals without a phagocytic defect such fucosyl-containing glycoconjugates are not as accessible and may be masked by other sugar residues in the oligosaccharide chain.

Lectin cytochemistry and freeze-fracture study of phagocytosis in the rat retina.

Lectin cytochemistry and freeze-fracture intramembrane morphology have been studied in adult rat retinas during various stages of the phagocytic cycle. Animals were perfused with aldehydes at various times during the first 6 hours of light and the retinas were removed. Chopped sections of tissue were stained with one of two lectin-peroxidase conjugates: ricinis communis agglutinin (RCA), which is specific for n-acetylgalactosamine and galactose, or wheat germ agglutinin (WGA), which is specific for n-acetylglucosamine and sialic acid. Other sections were frozen and prepared for freeze fracture. During the first hour of light onset, numerous phagosomes are already present in the retinal pigment epithelial (RPE) cytoplasm. The engulfment stages of phagocytosis whereby the RPE microvilli wrap around and enclose shed rod outer segment (ROS) tips are rarely observed at this time. Newly formed phagosomes are present in the apical RPE cytoplasm while older and more osmiophilic phagosomes are located deeper within the RPE cytoplasm. Between 2 and 4 hours of light onset fewer phagosomes are present in the RPE cytoplasm, but it is common to observe many ROS tips in various stages of microvillous engulfment. By 6 hours phagosomes are rarely present but some engulfed ROS tips continue to be observed. Both RCA and WGA stain RPE microvilli and plasma membranes of photoreceptor outer segments. WGA, in addition, labels some intradisc membranes of intact and also shed outer segments that are being engulfed but does not label disc membranes in phagosomes. At all times, phagosomes are rimmed by both lectin stains, but their internal contents are never stained. Lysosomes and multivesicular and residual bodies are labeled by both lectins. Freeze-fractured retinas show that as the ROS tips are engulfed, particle-free areas are present on disc P-face (cytoplasmic) membranes. Many newly formed phagosomes also display particle-free membrane areas and older phagosome membranes are completely devoid of particles. These observations show that the changes in WGA staining and intramembrane particle distribution on disc membranes occur in retinas during phagocytosis and suggest that some membrane component that is present for the initial phagocytic interaction may be rapidly degraded thereafter.

Surface-replica topography of retinal pigment epithelium during phagocytosis.

The initial step in retinal phagocytosis may be mediated by complementary recognition molecules on the pigment epithelial microvilli and outer segment membrane surfaces. These molecules may display a surface morphology which is related topographically to phagocytic events. In order to explore this, we have developed a method for replicating the membrane surfaces of rat pigment epithelial explants during phagocytosis of latex beads. The explants are fixed in a mixed aldehyde and osmium fixative and the basal surface glued to a coverslip. After dehydration and critical point drying, the tissue is replicated with platinum and carbon in a freeze-fracture apparatus using either rotary or unidirectional coating and the replicas are examined by transmission electron microscopy. The membrane surfaces of the pigment epithelial cells and their microvilli are studded with numerous surface particles varying in size from 20 to 50 nm that are closely packed and give a cobblestone appearance to the membrane topography. During the early stages of phagocytosis, microvillous processes appear to spread over the beads and form branched processes as they contact bead surfaces. During bead engulfment the microvilli have shortened into flattened sheets with interdigitating processes overlapping the beads. These uptake sites resemble Venus' fly-traps, which as they close over the beads create craters in the membrane surfaces of the pigment epithelium. The crater-like uptake sites appear to flatten out in the later stages and form flattened membrane domains which are surrounded by crenulated membrane that displays an irregular particulate morphology.

Synaptic membrane domains in photoreceptors of chick retina: a thin-section and a freeze-fracture study.

In this freeze-fracture study of synaptic terminals of chick photoreceptors, three regions of synaptic terminal plasmalemma can be distinguished on the basis of intramembrane characteristics. The first region is the synaptic vesicle fusion region in which rows of P-face depressions and E-face mounds are observed. In the absence of exocytotic figures this zone is relatively free of P-face particles and E-face pits. Adjacent to this, a second region is seen, rich in P-face particles and complementary E-face pits. This second region waxes and wanes in size during dark and light stimulation (Cooper and McLaughlin, 1982) and may correspond to similar expansions and contractions of synaptic plasmalemma induced by less physiological modes of stimulation, as observed in other synaptic terminals (Ceccarelli et al., 1979b; Model et al., 1975; Boyne and McLeod, 1979). During the waxing period, P-face particles and E-face pits are present in this membrane, and its expansion gives rise to diverticula of the synaptic terminal. During the waning period when the diverticula begin to disappear, aggregates of P-face particles and complementary patches of E-face pits are seen in the diverticular membrane. The third region, the nonsynaptic plasmalemma enclosing the terminal, contains a high density of P-face particles but does not contain E-face pits. Serial sections of vacuoles within the cytoplasm demonstrate that some vacuoles retain connections with this nonsynaptic plasmalemma. Vacuoles that are connected in this way are depleted of intramembrane particles. Such regions appear to represent separate domains within the photoreceptor terminal and are discussed in the context of membrane addition and retrieval.

Intramembrane changes in retinal pigment epithelial cell junctions of the dystrophic rat retina.

A progressive failure in phagocytosis by the retinal pigment epithelium (RPE) occurs in the Royal College of Surgeons rat with inherited retinal degeneration. Another change that can be attributed to a defect in the RPE is a breakdown in the blood-retinal barrier. RPE cell junctions, which form a part of this barrier, become permeable to extracellular tracer during the dystrophic process. We have used the freeze-fracture technique to study the structure of RPE cell junctions in normal and dystrophic retinas. In normal retinas, tight junctions between RPE cells consisted of 8 to 16 anastomosing strands on the cytoplasmic membrane leaflet (P-face) and a complementary pattern of grooves on the external membrane leaflet (E-face). Gap-junctional aggregates of hexagonally packed P-face particles and complementary E-face pits were enclosed within the tight junctional strands. In dystrophic retinas changes were first seen at postnatal day 21. Subtle breaks in P-face tight-junctional strands became more pronounced with time. Eventually the tight junctions appeared to unravel from the gap junctional aggregates, which became isolated and appeared to break off into patches of particle aggregates. The increased density of background particles in the membranes adjacent to disassembling junctions suggested that junctional elements were being removed by dispersal. Endocytosis of junctional elements was observed in both dystrophic and control retinas but may be accelerated in the dystrophic retina. In the late stages of the dystrophy some RPE cell junctions appeared to have proliferated and occupied extensive areas of the RPE membrane.

Freeze-fracture study of photoreceptor outer segments and pigment epithelium in dystrophic and normal retinas.

The intramembrane organization of outer segment (OS) membranes and pigment epithelial (PE) microvilli has been studied in rats (10-17 postnatal days) with inherited retinal degeneration (RCS) and in normal retinas from genetically controlled rats (RCS-rdy+). The OS plasma membranes of both dystrophic and normal retinas are characterized by large particles surrounding circular, particle-free zones on the P-faces (cytoplasmic leaflets) and a sparse distribution of particles on the E-faces (external leaflets). No regional differences in particle distribution are observed in either basal or distal plasma membrane regions. Outer segment disc membranes are characterized by large, densely packed P-face particles and ridged E-faces with very few particles. Small, particle-free patches of membrane are present in the basal disc P-faces of both normal and dystrophic retinas, which Andrews and Cohen ("79) have described as characteristic of newly added disc membrane. In dystrophic retinas, larger, particle-free domains are observed in the distal disc membranes (P-faces) and accumulating membranous debris. In older retinas, which have accumulated more debris, the particle-free domains occupy vast areas of the membrane faces and it is not possible to identify these membranes as belonging to either discs of plasma membranes. No comparable areas of particle-free membrane are observed in the distal discs and OS plasma membranes of normal retinas. Pigment epithelial microvillus membranes are characterized by an intermixture of large and medium-sized particles surrounding irregular particle-free areas, but no differences between normal and dystrophic PE membranes are observed. The changes in particle distribution observed in the dystrophic retinas suggests that the intramembrane molecular composition of older disc membranes has become altered or rearranged as the OS membranes accumulate as debris.

A freeze-fracture study of synaptic junction development in the superficial layers of the chick optic tectum.

Synaptogenesis in the superficial layers of the rostral pole of the chick optic tectum has been studied using freeze-fracture techniques. The developmental sequence of intramembrane organization at synaptic junctions involves the accumulation and assembly of intramembrane particles into aggregates characteristic of the mature junctions. By embryonic day seven, areas of loosely-arranged clusters of medium-sized particles are observed on the cytoplasmic membrane leaflets (P-faces) of developing neurites. These clusters are characteristic of the intramembrane organization at presynaptic active zones. At later stages, small pits, characteristic of vesicle fusion sites, are observed interspersed among such P-face particle clusters. Complementary intramembrane specializations are also present on the external leaflets (E-faces) of presynaptic membranes at the active zones. Small solitary aggregates of large-sized particles on the E-faces of neurite plasma membranes are also seen at early embryonic stages. As development progresses, these aggregates increase in size and packing density and occupy large oval domains in postsynaptic membranes. These intramembrane specializations may represent the postsynaptic active zones of asymmetric synapses. Another type of intramembrane specialization, observed during the third week of incubation, is characterized by aggregates of small- and medium-sized particles on the P-face of postsynaptic membranes and is often seen directly apposed to the E-face of a presynaptic terminal. This type of intramembrane specialization may represent the postsynaptic active zone region at symmetrical synaptic contacts.